Cardiovascular diseases are among the leading causes of death in North America. Currently, there are no effective treatment options for directly repairing the damaged myocardial tissue. Therefore, cell-based therapies utilizing cardiomyocytes generated from stem cells to replace necrotic tissue will be a promising approach. However, the molecular mechanisms regulating stem cell differentiation into cardiomyocytes are not fully understood. Since GATA-4 is one of the primary regulators of cradiomyogenesis, we investigated the molecular basis of GATA-4 expression during the early stages of stem cell differentiation. Using chromatin immunoprecipitation, we have observed the direct involvement of p300 in GATA-4 gene expression. We have also examined the importance ofhHistone acetylation and acetyltransferase activity on GATA-4 expression during the early stage of cardiomyogensis using the histone deactylase inhibitor Valproic Acid and the acetyltransferase inhibitor Curcumin respectively.

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Le cœur est un organe central au fonctionnement du système cardiovasculaire. Il est physiologiquement compartimenté et est constitué de cellules spécialisées qui régulent les impulsions électriques ainsi que la contraction du myocarde. Le cœur adapte le flux sanguin en fonction des besoins du corps. En condition pathologique, le cœur recourt toutefois à des mécanismes compensatoires. Au niveau physiologique, la compensation s’observe par l’hypertrophie des cardiomyocytes qui, bien que bénéfique à court terme, exacerbe à long terme la fonction cardiaque. L’activation des « mitogen activated protein kinases » (MAPK) contribue autant au maintien de la fonction physiologique qu’à la détérioration pathologique du myocarde et serait également une cause de l’hypertrophie observée. Parmi les 5 groupes de MAPK connues, la MAPK p38 est formée de 4 isoformes dont les sérine/thréonine kinases p38α et p38γ sont exprimées de façon prédominante dans le cœur. Les p38 partagent les mêmes activateurs, mais leurs effecteurs diffèrent. Bien que le rôle de p38α semble impliqué dans l’aggravement des troubles cardiaques, celui de p38γ ne semble pas redondant à p38α et demeure incompris. Cette isoforme possède un motif de liaison aux domaines PDZ, unique chez les MAP kinases. Également, chez les cellules cardiaques, elle transloque au noyau en condition de stress. Le but de l’étude ici est de comprendre le rôle de p38γ et de ses motifs uniques sur la structure et la taille des cardiomyocytes. Afin de répondre au but de l’étude, plusieurs mutants adénoviraux de p38 ont été conçus. Un des mutants ne possède pas le motif de liaison aux domaines PDZ, deux autres contrôlent la localisation cellulaire soit au noyau, soit au cytoplasme, et un autre mutant est muté au site de phosphorylation. Des cardiomyocytes en culture ont été infectés par les différents mutants en présence de leur activateur en amont ou de la β-galactosidase. Les réseaux d’α-actinine, ainsi que la taille des cardiomyocytes, ont été observés par microscopie. Les observations effectuées montrent que p38γ entraîne une désorganisation des réseaux d’α-actinine lorsqu’il est phosphorylé. Également, il facilite l’hypertrophie des cardiomyocytes en présence de son activateur s’il est forcé hors du noyau ou en l’absence de son motif de liaison aux domaines PDZ. En conclusion, les résultats obtenus suggèrent que p38γ exerce bel et bien un rôle dans le maintien structural des cardiomyocytes par l’intermédiaire de l’α-actinine.

Résumé: L’autophagie est un processus essentiel au maintien de l’homéostasie cellulaire. Elle permet de dégrader et recycler aussi bien des organelles entières que des composants cytoplasmiques non fonctionnels. De plus, l’augmentation d’autophagie en condition de stress constitue une réponse adaptative favorisant la survie cellulaire. Chez les cardiomyocytes, l’autophagie en condition basale est indispensable au renouvellement, entre autres, des mitochondries et des protéines formant les sarcomères. De plus, les stress tels l’ischémie cardiaque ou la carence en nutriments induisent une augmentation de l’autophagie protectrice. Dans certaines conditions extrêmes, il a été suggéré qu’un surcroît d’autophagie puisse toutefois exacerber la pathologie cardiaque en provoquant la mort des cardiomyocytes. Considérant l’importance de ce processus dans la physiopathologie cardiaque, l’identification des mécanismes signalétiques régulant l’autophagie chez les cardiomyocytes a été le sujet de recherches intenses. À cet effet, l’activation des Mitogen-Activated Protein Kinase (MAPK) a été démontrée pour réguler, avec d’autres voies signalétiques, l’autophagie et l’apoptose des cardiomyocytes. Il est donc probable que les Dual-Specificity Phosphatase (DUSP), enzymes clés contrôlant l’activité des MAPK, participent aussi à la régulation de l’autophagie. Afin de vérifier cette hypothèse, nous avons induit l’autophagie chez des cardiomyocytes isolés de rats nouveau-nés en culture. L’analyse de marqueurs d’autophagie par immunobuvardage démontre que l’activation des MAPK ERK1/2 et p38 corrèle avec l’activité autophagique chez les cardiomyocytes. Dans ces conditions, la diminution d’expression de la majorité des ARNm encodant les différentes DUSP retrouvées chez les cardiomyocytes contraste de façon marquée avec l’augmentation d’expression de l’ARNm Dusp5. De plus, nous avons démontré par une étude de gain de fonction que l’activation soutenue de p38 par surexpression d’un mutant MKK6 constitutivement actif stimule l’autophagie chez les cardiomyocytes. De façon surprenante, la perte de fonction de p38 obtenue par surexpression d’un mutant p38 dominant négatif n’altère en rien la réponse autophagique initiatrice dans notre modèle in vitro. Nos résultats suggèrent que les DUSP puissent réguler, via leurs actions sur les MAPK, d’importantes étapes du processus autophagique chez les cardiomyocytes. / Abstract: Autophagy is a process essential to the maintenance of cellular homeostasis. It helps degrade and recycle whole organelles and nonfunctional cytoplasmic components. In addition, the adaptative up regulation of autophagy in stress condition promotes cell survival. In cardiomyocytes basal autophagy is essential to the renewal of, among others, mitochondria and proteins forming sarcomeres. In addition, stresses such as ischemic heart or nutrient deficiency induce an increase in protective autophagy. In extreme conditions, it has been suggested that autophagy may exacerbate cardiac disease causing the death of cardiomyocytes. Considering the importance of this process in cardiac pathophysiology, identify ing safety mechanisms regulating autophagy in cardiomyocytes has been the subject of intense research. To this end, activation of mitogen-activated protein kinase (MAPK) has been demonstrated to regulate, with other signaling pathways, autophagy and cardiomyocyte apoptosis. It is therefore likely that Dual-Specificity Phosphatases (DUSPs), key enzymes that control the activity of MAPKs, also participate in the regulation of autophagy. To test this hypothesis, we have induced autophagy in isolated cardiomyocytes of newborn rats in culture. Analysis of autophagy markers by immunoblotting demonstrated that the activation of MAPKs ERK1/2 and p38 correlates with autophagic activity in cardiomyocytes. Under these conditions, the decrease in expression of the majority of mRNAs encoding different DUSPs found in cardiomyocytes contrast sharply with the increase mRNA expression of Dusp5. Furthermore, we demonstrated by again of function study that sustained activation of p38 by overexpression of a constitutively active MKK6 mutant stimulates autophagy in cardiomyocytes. Surprisingly, the loss of p38 function obtained by overexpression of a dominant negative p38 mutant does not affect the autophagic response in our in vitro model, but increases the lipidation of autophagosomes marker LC3. Our results suggest that DUSPs can regulate, through their actions on MAPKs, important stages of autophagy in cardiomyocytes.

MSc (Med), University of the Witwatersrand, Faculty of Health Sciences / Background: Males have a higher prevalence of cardiovascular diseases compared to premenopausal women. However, postmenopausal women are at equal risk to men. It has therefore been suggested that estrogen is cardioprotective. Although the exact mechanisms of the purported cardioprotective effects of estrogen are unknown, estrogen administration has been reported to suppress beta-adrenergic receptor up-regulation in ovariectomized female rats. As beta-adrenergic activation induces cardiomyocyte apoptosis and necrosis, and hence adverse cardiac remodelling and heart failure, I aimed to determine whether the extent of beta-adrenergic induced apoptosis and necrosis differs between males and females. Methods: 27 male Wistar rats were assigned to one of two groups: ISO M (n=14) receiving a beta-adrenergic receptor agonist, isoproterenol (0.02mg/kg) and CON M (n=13) receiving vehicle (saline, 0.2ml). 29 female Wistar rats were assigned to one of two groups: ISO F (n=15) receiving a beta-adrenergic receptor agonist, isoproterenol (0.02mg/kg) and CON F (n=14) receiving vehicle. Isoproterenol and saline were administered by means of daily subcutaneous injections for 5 days. On the 5th day, cardiac geometry and function were assessed before and after ISO or saline administration using echocardiography. Rats were then terminated under anaesthesia within 30 minutes of ISO (or vehicle) administration and blood samples collected for the determination of serum estrogen concentration (ELISA). Female rats were terminated in proestrus which corresponds to peak estrogen concentrations. Cardiac myocyte apoptosis was assessed histologically using the DeadEndTM Colorimetric TUNEL system (Promega, Madison, WI, USA). The number of apoptotic cardiomyocyte nuclei was expressed as a percentage of the total number of cardiomyocyte nuclei per slide (heamotoxylin and eosin stain). Necrosis and fibrosis (pathological score) were assessed by assigning a pathological score to sections stained for fibrosis (van Gieson). Groups were iii compared using two-way (gender and regimen; and including repeated measures for echocardiography data) ANOVA followed by the Tukey-Kramer post hoc test. Results: As expected estrogen concentrations were higher in female compared to male rats (mean±SEM, pg.ml-1; ISO M: 7.04±1.41; CON M: 7.14±0.53; ISO F: 23.00±3.47; CON F: 19.31±3.66; p<0.01). Five days of ISO or saline administration had no effect on cardiac function or geometry in either the male or the female rats. Inotropic effects (increased heart rate and cardiac function) were observed in response to acute ISO administration in both male and female rats. The female rats had slower heart rates (p<0.05) and showed a greater heart rate response to acute ISO administration than the male rats (p<0.05). But the acute ISO induced increments in cardiac function were similar between genders. Five days of ISO administration induced cardiomyocyte apoptosis in male rats but not in female rats (mean±SEM, % ; ISO M: 0.086±0.013; CON M: 0.030±0.004; ISO F: 0.053±0.004; CON F: 0.041±0.007; p<0.05). Furthermore, 5 days of ISO administration induced cardiomyocyte necrosis in male rats but not in female rats (mean±SEM, pathological score; ISO M: 1.21±0.21, CON M: 0.46±0.14, ISO F: 0.50±0.11, CON F: 0.68±0.12, p<0.01). Conclusion: Male rats are more susceptible than female rats to beta-adrenergic induced cardiomyocyte apoptosis and necrosis. The protective effects of estrogen against the adverse effects of beta-adrenergic activation on the heart, may explain the lower risk of cardiovascular disease in premenopausal women compare to men; however, the possible role of progesterone cannot be ignored.

The cellular microenvironment consists of soluble and insoluble factors that provide signals that dictate cell behavior and cell fate. Limited characterization has hindered our ability to mimic the physiological or pathophysiological environment. While stem cells have vast promise in the areas of regenerative medicine and disease therapy, harnessing this potential remains elusive due to our limited understanding of differentiation mechanisms. Similarly, many in vitro cardiac disease models lack the critical structure- function relationships of healthy and diseased cardiac tissue. The goal of this work is to induce cardiomyogenesis and pathogenesis in vitro by recapitulating features of the native microenvironment during development and disease. / Engineering and Applied Sciences

Oxidative stress is known to contribute to many forms of heart disease. Oxidants such as H₂O₂ can cause hypertrophy of cardiomyocytes (CMCs). Heart fibroblasts (HFs) also contribute to oxidant-induced heart disease by disordering the extracellular matrix and causing fibrosis. Since both of these cells encounter the same stressors in vivo, we examined the signaling pathways involved in responding to oxidative stress in both cell types. We have established the EGF Receptor, Src and matrix metalloproteinases (MMPs) as key regulators of oxidant-mediated phosphorylation of the MAPKs ERK1/2 and JNKs but not p38 in CMCs and HFs. We used oligonucleotide microarrays to examine the differences in global gene expression after H₂O₂ treatment in CMCs and HFs. Twenty-four hours after treatment, significant numbers of upregulated genes could be classified as being related to antioxidant or detoxification responses in both cell types. This trend lead us to examine the role of activation of promoters containing the Antioxidant Response Element (ARE) in the reaction of CMCs to H₂O₂. We have shown that H₂O₂ activates the ARE in CMCs in a manner that is dependant on the transcription factor Nf-E2 related factor 2 (Nrf2). ARE activation by H₂O₂ seems to induce cytoprotection. CMCs pretreated with H₂O₂ showed significantly less activation of caspase-3 when exposed to another oxidant, Doxorubicin. Overexpression of Nrf2 mediates this cytoprotection, possibly by protecting the cells from caspase-independent cell death. Although ARE-dependant genes were upregulated in the presence of excess Nrf2, two contractile proteins were repressed, suggesting that Nrf2 overexpression may have unknown side-effects in CMCs. We also studied the activation mechanism of Nrf2 in CMCs. Nrf2 protein levels increased after 10 min of exposure to 100 μM H₂O₂ and peaked at about 1 hr. Pharmacological and genetic inhibition of the PI3-Kinase pathway blocked AREluciferase activity in these cells. The PI3-Kinase inhibitor LY294002 also blocked Nrf2 protein accumulation, but not nuclear translocation. Here I present evidence that Nrf2 accumulation after H₂O₂ exposure is due to PI3-Kinase-mediated translational regulation. Since phosphorylation of translation initiation factors eIF4E and eIF2alpha are both inhibited by LY294002, Nrf2 translation initiation may be through non-5’ cap-mediated means.

In adult cardiomyocytes, the established mechanism of excitation-contraction coupling is calcium-induced calcium release (CICR) mediated by L-type Ca2+ channels (Cav1.2). Briefly, membrane depolarization opens voltage-gated Cav1.2 to allow for the influx of extracellular Ca2+ into the cytosol. This small sarcolemmal (SL) Ca2+ influx is necessary for triggering a larger release of Ca2+ from the intracellular Ca2+ storage site, the sarcoplasmic reticulum (SR), through the SR Ca2+ release channel also known as the ryanodine receptor (RyR). RyR-mediated release of SR Ca2+ effectively raises the cytosolic free Ca2+ concentration, allowing for Ca2+ binding to troponin C on the troponin-tropomysin complex, leading to cross-bridge formation and cell contraction. However, previous functional data suggests an additional CICR modality involving reverse mode Na+-Ca2+ exchanger (NCX) activity also exists in neonate cardiomyocytes. To further our understanding of how CICR changes occur during development, we investigated the spatial arrangement of caveolin-3 (cav-3), the principle structural protein of small membrane invaginations named caveolae, and NCX in developing rabbit ventricular myocytes. Using traditional as well as novel image processing and analysis techniques, both qualitative and quantitative findings firmly establish the highly robust and organized nature of NCX and cav-3 distributions during development. Specifically, our results show that NCX and cav-3 are distributed on the peripheral membrane as discrete clusters and are not highly colocalized throughout development. 3D distance analysis revealed that NCX and cav-3 clusters are organized with a distinct longitudinal and transverse periodicity of 1-1.5 μm and that NCX and cav-3 cluster have a pronounced tendency to be mutually exclusive on the cell periphery. Although these findings do not support the original hypothesis that caveolae is the structuring element for a restricted microdomain facilitating NCX-CICR, our results cannot rule out the existence of such microdomain organized by other anchoring proteins. The developmentally stable distributions of NCX and cav-3 imply that the observed developmental CICR changes are achieved by the spatial re-organization of other protein partners of NCX or non-spatial modifications. In addition, the newly developed image processing and analysis techniques can have wide applicability to the investigations on the spatial distribution of other proteins and cellular structures.

Phosphodiesterases (PDEs) are the enzymes responsible for the hydrolysis of cyclic nucleotides including cAMP and cGMP. We recently discovered that natriuretic peptides elicit effects in the atrial myocardium via a PDE dependant pathway; however, the role(s) of specific PDE subtypes in atrial myocytes are not clear. Thus, I studied the effects of PDE selective blockers on mouse atrial action potentials (APs) and L-type Ca2+ currents (ICa,L). AP duration (APD) was significantly increased in the presence of IBMX (inhibits all PDEs) as well as EHNA (PDE2 inhibitor) and rolipram (PDE4 inhibitor). The PDE 3 inhibitor milrinone had no effect on APD. Applying milrinone and rolipram (PDE3/PDE4 inhibition) or EHNA, milrinone, and rolipram (PDE2/ PDE3/PDE4 inhibition) in combination prolonged APD as effectively as IBMX. A similar pattern of results was obtained for atrial ICa,L. These data provide novel insight into the unique effects of PDE inhibitors in atrial myocytes

Diabetic cardiomyopathy is defined as changes in the structure and function of the myocardium that occur in diabetic patients in the absence of other cardiovascular risk factors. Our laboratory has shown that conjugated linoleic acid (CLA - a naturally-occurring polyunsaturated fatty acid with multiple health benefits) prevents endothelin-1-induced myocyte hypertrophy in vitro, as well as cardiac hypertrophy in vivo using a rodent model of spontaneously hypertensive heart failure. These cardioprotective effects of CLA were mediated through activation of peroxisome proliferator activated receptors (PPAR isomers α and γ) and stimulation of diacylglycerol kinase ζ (DGKζ). Thus, the aims of this study were to (i) determine the effect of CLA on hyperglycemia-induced structural and functional abnormalities of cardiomyocytes, and (ii) assess the role of PPAR-γ and DGK. High glucose treatment induced hypertrophy of primary adult cardiomyocytes, as indicated by augmented cell size and protein synthesis compared to untreated cardiomyocytes. The hyperglycemia-induced hypertrophy was attenuated by pretreatment with CLA (30 µM). The ability of CLA to prevent hyperglycemia-induced hypertrophy was suppressed by GW9662 (1 µM) and R59022 (10 μM), pharmacological inhibitors of PPAR-γ and DGK, respectively. In addition to structural abnormalities, high glucose impaired contractile function of adult cardiomyocytes as measured by maximal velocity of shortening, maximal velocity of relengthening, and peak shortening. Hyperglycemia-induced contractile dysfunction was likewise prevented by pretreatment with CLA (30 µM). Collectively, these findings support the idea that hyperglycemia is an independent risk factor for the development of diabetic cardiomyopathy. Hypertrophy and contractile dysfunction elicited by high glucose were prevented by CLA. The antihypertrophic actions of CLA are mediated, at least in part, by activation of PPAR-γ and DGK.